Automated Online-Sampling Multidimensional Liquid Chromatography with Feedback-Control Capability as a Framework for Real-Time Monitoring of mAb Critical Quality Attributes in Multiple Bioreactors.

Real-time monitoring of biopharmaceutical reactors is becoming increasingly important as the processes become more complex. During the continuous manufacturing of monoclonal antibodies (mAbs), the desired mAb product is continually created and collected over a 30 day process, where there can be changes in quality over that time. Liquid chromatography (LC) is the workhorse instrumentation capable of measuring mAb concentration as well as quality attributes such as aggregation, charge variants, oxidation, etc.

HDX-MS and MD Simulations Provide Evidence for Stabilization of the IgG1-FcγRIa (CD64a) Immune Complex Through Intermolecular Glycoprotein Bonds.

Previous reports present different models for the stabilization of the Fc-FcγRI immune complex. Although accord exists on the importance of L235 in IgG1 and some hydrophobic contacts for complex stabilization, discord exists regarding the existence of stabilizing glycoprotein contacts between glycans of IgG1 and a conserved FG-loop (MGKHRY) of FcγRIa. Complexes formed from the FcγRIa receptor and IgG1s containing biantennary glycans with N-acetylglucosamine, galactose, and α2,6-N-acetylneuraminic terminations were measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS), classified for dissimilarity with Welch's ANOVA and Games-Howell post hoc procedures, and modeled with molecular dynamics (MD) simulations.

Cell Free Remodeling of Glycosylation of Antibodies.

The N-glycosylation profile of a monoclonal antibody (mAb) is a critical quality attribute in relation to its therapeutic application. The control of this profile during biomanufacture is difficult because of the multiple parameters that affect the glycosylation metabolism within the cell and the environment in which the cell is grown. One of the approaches that can be used to produce a preferred glycan profile or a single glycoform is through chemoenzymatic remodeling during the isolation of a mAb.

Strategic feeding of NS0 and CHO cell cultures to control glycan profiles and immunogenic epitopes of monoclonal antibodies.

The control of glycosylation profiles is essential to the consistent manufacture of therapeutic monoclonal antibodies that may be produced from a variety of cell lines including CHO and NS0. Of particular concern is the potential for generating non-human epitopes such as N-glycolylneuraminic acid (Neu5Gc) and Galα1-3 Gal that may be immunogenic. We have looked at the effects of a commonly used media supplements of manganese, galactose and uridine (MGU) on Mab production from CHO and NS0 cells in enhancing galactosylation and sialylation as well as the generation of these non-human glycan epitopes.

Solid-Phase Enzymatic Remodeling Produces High Yields of Single Glycoform Antibodies.

Antibodies are synthesized in mammalian cell culture as heterogeneous mixtures of glycoforms. Production of single glycoforms remains a challenge despite their value as therapeutics. The authors report a method of sequential enzymatic-based changes to antibodies while immobilized on an affinity column.

A systematic study of glycopeptide esterification for the semi-quantitative determination of sialylation in antibodies.

Rationale: In the expression of recombinant proteins, an important parameter to control or influence is their level of sialylation. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric (MS) methods tend to either underestimate (positive mode) or overestimate (negative mode) the content of sialylated vs. neutral glycans in glycoproteins.

Low glucose depletes glycan precursors, reduces site occupancy and galactosylation of a monoclonal antibody in CHO cell culture.

Controlled feeding of glucose has been employed previously to enhance the productivity of recombinant glycoproteins but there is a concern that low concentrations of glucose could limit the synthesis of precursors of glycosylation. Here we investigate the effect of glucose depletion on the metabolism, productivity and glycosylation of a chimeric human-llama monoclonal antibody secreted by CHO cells. The cells were inoculated into media containing varying concentrations of glucose.

Mathematical model of the rate-limiting steps for retrovirus-mediated gene transfer into mammalian cells.

A quantitative understanding of the process of retrovirus-mediated gene transfer into mammalian cells should assist the design and optimization of transduction protocols. We present a mathematical model of the process that incorporates the essential rate-limiting transduction steps including diffusion, convection and decay of viral vectors, their binding at the cell surface and entry into the cell cytoplasm, reverse transcription of uncoated RNA to form DNA intermediates, transport of the latter through the cytosol to the cell nucleus and, finally, nuclear import and integration of the delivered DNA into the target cell genome. Cell and virus population balances are used to account for the kinetics of multiple vector infections which influence the transduction efficiency and govern the integrated copy number.